Glycoside metabolites of oospora virescens wallr fungus

ABSTRACT

Eight Glycoside Metabolite compounds are produced by the Oospora virescens (Link) Wallr fungus in glucose-organic decoction media which can be separated from the growth media and from each other. These compounds are effective bacterial and antimycotic agents.

United States Patent [191 Bellavita GLYCOSIDE METABOLITES OF OOSPORAVIRESCENS WALLR FUNGUS [76] Inventor: Nera Cagnoli Bellavita, Via XXSettembre 2, Perugia, Italy [22] .Filed: Aug. 23, 1973 21 Appl. No.:390,730

Related U.S. Application Data [62] Division of Ser. No. 105,477, Jan.11, 1971.

[52] U.S. Cl. 195/81, 195/31 [51] Int. Cl C12b 1/00 [58] Field of Search195/35, 81, 100, 36, 37

[56] References Cited OTHER PUBLICATIONS Bellavita, et al., GazzettaChimica ltaliana Vol. 98, p. 1354-1363.

[ Dec. 17, 1974 Primary ExaminerA. Louis Monacell AssistantExaminerThomas G. Wiseman Attorney, Agent, or Firm-John .l. Lipari 1 5 7ABSTRACT 6 Claims, 9 Drawing Figures PATENTED DEC] 7 I974 SHEET 5 or 5THIN LAYER CH ROMATOGRAM GLYCOSIDE METABOLITES OF OOSPORA VIRESCENSWALLR FUNGUS This is a divisional of copending application, Ser. No.105,477, filed Jan. 11,1971.

BACKGROUND OF THE INVENTION The Oaspora virescens (Link) Wallr fungusforms glycoside metabolites. 'Two of these glycosides have beendescribed in Gazzetta Chimica Italiana," vol. 98 p. 1354 1369, 1969.

SUMMARY OF THE INVENTION wherein R has the meaning given above and Y isCHO or CH OH with the proviso that when Y is -CH OH, R is hydrogen, and

crnoH o-crn The mixture of metabolites produced in the media can beseparated from the cultlure by lyophilizing the culture and extractingwith ethanol, and separated from each other by chromatographic columntechniques according to the processes described more fully hereinbelow.In addition, certain of the glycoside metabolites can be prepared fromothers of these compounds. vThe mixture of metabolites as well as theindividual glycosides are useful antimycotic agents. In addition,various of the glycoside compounds are useful antibacterial agents.

Therefore it is a principle object of the present invention to providenew compositions of matter and methods of preparing them.

It is another object to provide a mixture of glycosides produced by theOaspora virescens (Link) Wallr fungus.

It is still another object to provide a method of separating suchglycosides from each other.

It is another object to provide a method of inhibiting the growth offungi.

It is another object to provide a method of inhibiting the growth ofbacteria.

Further objects will become apparent from the following detaileddescription thereof.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 8 are infrared spectra ofcompounds I through VIII. FIG. 9 is a picture of two developed thinlayer chromatograms of compounds I through VIII.

DETAILED DESCRIPTION OF THE INVENTION Compound I CHO K" II II HOI OH HCompound II Compound I11 Compound V Compound IV CHzOH 0 Compound VICompound VII Compound VIII ,Kgial According to the present process, aglycosideproducing strain of Oospora virescens (Link) Wallr in anaqueous glucose-organic decoction media is cultivated at a temperaturefrom 10 to 25 C. in a stationary culture for a period of at least 10days.

' The glycoside-producing strain of the Oospora virescens (Link) Wallrare deuteromycetes of the Moniliaceae family, isolated from the deadbranches of the mulberry bush. A colony is selected in agar malt from acolony differentiated from others by a more rapid and intensesporulation. In organic solid or liquid media based upon malt and onvegetable decoctions with or without glucose, such as wheat kernels,corn kernels, oat kernels, carrots, kidney bean seeds and the like, thefungus colonies are round with a slightly sinuous out line, at firstflatted-and white, then becoming raised; they change with the degree ofdevelopment to become green. They become dark green and velvety withtime.

The vegetative mycelium consists of hyaline, branched,

septate hyphae, about 2.5 microns wide, which support numerousconidiophores bearing abundant catenulate spores, produced in basipetalsuccession in very long, flexible chains. The conidiophores are simple,hyaline, erect or bent, about 30 to 40 microns long, slightly swollen atthe base and narrowed towards the tip. The spores are fusiform, smooth,about 7 to 8 microns long and about 2.5 to 3 microns wide, having agreen or olive green color.

The temperature of cultivation can be from 10 to 25 C., preferably from23 to 25 C. Growth of the fungus is slow at C. and above about C. nogrowth occurs.

The culture must be stationary in order for the glycosides of theinvention to be formed.

The organic media found suitable for the production of the glycosides ofthe invention includes aqueous decoctions of malt, glucose-yeast,glucose-carrot, glucose-wheat kernels, glucose-corn kernels,glucosekidney bean seeds, glucose-pea seeds and glucose-lentil seeds.The preferred decoction is glucose-carrot.

The pH of the media is important, particularly with respect to theformation of compounds I and 1V. Preferably the pH of the culture mediais basic, most preferably within the range 8.6 to 8.8. This can be doneby adding a suitable base, such as an alkali metal or ammonium hydroxideto the media. Sodium hydroxide is preferred.

The cultures as prepared above are lyophilized and extracted withethanol. The ethanol extract is then cooled slowly, when the metal saltsof acid compounds VII and Vlll form a precipitate. The resultant mixtureis filtered and the filtrate dried and charged to a chromatographiccolumn to separate the remaining compounds from each other, usingconventional techniques.

The compounds are best separated using a mixture of chloroform-methanol,but other known solvents can also be employed. The chromatogram isdeveloped in conventional manner using sulphuric acid.

The precipitated salts are treated with a suitable acid, such as dilutehydrochloric acid, and separating from each other on a chromatographiccolumn using chloroform-methanol as above.

In addition to the procedures described above, certain of the glycosidemetabolites described herein can also be prepared from others of thesecompounds. For example, compound I can be prepared from compound V bytrytilation, acetylation, detrytilation, oxidoelimination with theDoerings, reagent, and subsequent deacetylation with sodium methoxide inmethanom. Compound l can also be prepared by reducing the compound Vllwith a reducing agent, such as lithium aluminum hydride, to form thecompound V and treating as above to form compound I.

The compound N can be prepared in similar manner from the compound VIand by similar reduction of compound VIII.

The compounds of the invention show a wide spectrum of activity againstparasitic fungi, both of human beings and animals, in vitro and in vivo.In particular the compounds l and IV are of interest in that they havedemonstrated very low toxicity in mice, the LD,,,, of l in mice was 250mg/Kg subcutaneous, the LD of IV in mice was 250 mg/Kg subcutaneous. TheLD of ethanolic extract was 500 mg/Kg subcutaneous. The compounds of theinvention can be administered in the form of a powder, tablet, tinctureor ointment containing one or more the compounds of the invention asactive ingredient, together with conventional fillers and carriers.Suitable ointments can be prepared, for example, with excipient lanolinor fatty acids, such as undecylic acid or caprylic acid as the carrier.Powders and tablets can also be prepared in conventional manner. Forexample, a typical tablet can contain 70 mg of the active compound ofthe invention, 25 mg of ascorbic acid and 3 mg of potassiummetabisulfite.

Several of the compounds of the invention are particularly effectiveagainst particular fungi. For example, compound I is effective againstcutaneous mycosis from dermatophytes such as alopecia, eczema, herpes,ringworm, favus and histoplasmosis. Compound IV is of particularinterest against infections such as candidiasis or moniliasis andcryptococcosis, (above all cryptococcus Neoformans). The lattercompounds are most particularly of interest since they are effectiveagainst the pathogenic yeast Candida albicans, also known as Moniliaalbicans, which has resisted all treatment to date. These compounds arealso effective to inhibit the growth of bacteria, both of theGram-positive and the Gram-negative varieties.

The invention will be further illustrated by the following examples,butit is to be understood that the invention is not meant to be limitedto the details described therein. ln the examples, all percentages areby weight.

In preparing the cultures, the growth media were charged to one literRoux bottles and sterilized at a pressure of 24 psi for 3 to 4 minutesand then inoculated with 2 ml of a sterile, aqueous suspension ofabundant spores prepared from well sporulated, 20 to 40 day-old culturesof Oaspora virescens (Link) Wallr on oat-agar slants. The fungus isgrown in stationary culture in daylight, maintaining the temperaturefrom 23 to 25 C.

EXAMPLE I Three portions each of glucose-carrot and glucosecorn kernelsmedia after sterilization were adjusted to pH 5, 7 and 8.6 respectively,then inoculated and cultivated. After incubation for 20 to 30 days, themedia having pH 5 were found to have assimilated glucose slowly and thepH increased toward neutral values, whereas the assimilation of glucosein the other portions was much more rapid and the reaction mediumincreased toward pH 9.

After incubation of the media having pH 8.6 for 15 days, compounds II,V, VI, VII and VIII were produced; after 30 days, compounds 1, ill andIV were also produced. Maximum amounts of compounds I and IV wereproduced in the glucose-carrot decoction having pH 8.6-8.8.

EXAMPLE 2 Production of the compounds I to Vlll was compared in avariety of cultivation media as follows:

l) malt extract (powder), 15 gm/l 6 2) glucose (2%) yeast extract, 3grn/l 7.8 3) glucose (2%) carrot 5.5 4) glucose (2%) wheat kernels 5.85) glucose (2%) corn kernels 5.5 6) glucose (2%) kidney bean seeds 6 7)glucose (2%) a seeds 5.8 8) glucose (2%) entil seeds 6 9) glucose (2%)potato 5.5

The above media were sterilized, inoculated and cultivated as in ExampleI.

The growth and sporulation of the fungus was vigorous and abundant onmalt, glucose-carrot, glucosewheat kernels and glucosecorn kernels.

The growth was good but with poor sporulation on glucose-yeast,glucose-kidney bean seeds,'glucose-pea seeds, glucose-lentil seeds andglucose-potato media.

The compounds 11, V, VI, VII and VIII were produced in all of the abovemedia within 10 to 30 days of growth.

EXAMPLE 3 This example demonstrates that other media tested will notproduce the compounds of the invention and is given for purposes ofcomparison.

1. Czapeks modified liquid, prepared by admixing 1 gram potassiumhydrogen phosphate, 0.5 gram magnesium sulfate (MgSO, .7I-I O), 0.5 grampotassium chloride, 0.01 gram ferrous sulfate, 2 grams sodium nitrate,20 grams glucose and adding deionized water to make up 1 liter involume. This media has a pH of 6- 6.5.

2. Veindelings liquid, prepared by admixing 2 grams NH OCO(C1-IOI-1)COON1-1 1 gram potassium dihydrogen phosphate, 1 gram MgSO, H 0, 25grams glucose and deionized water to make up 1 liter in volume. Thismedia had a pH of 4.5 5.

. carrot decoction (250 gm./l), pH 6.

. wheat kernel decoction (140 gms/l), pH 6.

. corn kernel decoction (200 gms/l), pH 6.4.

. kidney bean seed de'coction (100 gms/l), pH 6.2. pea seed decoction(100 gms/l), pH 6.

. lentil seed decoction (100 gms/l), pH 6.

.potato decoction (200 gms/l), pH 5.5.

The above media were inoculated and cultivated as in Example 1.

The fungus grew poorly in media (1) and (2), but no sporulation wasnoted and none of the compounds I to VIII were produced.

The fungus grew more or less abundantly and sporulation was good tomoderate for media (3) to (9); however, none of the compounds 1 to VIIIwere produced after 30 days.

EXAMPLE 4 A medium for growth of Oaspora virescens (Link) Wallr funguscultures was prepared with carrot decoction in deionized water (250gms/l) and sterilized at a temperature of about 120 C. for 2-3 minutes.The decoction was filtered through cotton wool, glucose added, 2% of thetotal, and 300 ml of the resultant medium charged to three liter Lepinbottles sterilized at a temperature of 120 C. for 3-4 minutes. Aftercooling, the medium was adjusted to pH 8.6-8.8 with sterile sodiumhydroxide solution (1N) and inoculated with 5 ml of a sterile aqueoussuspension of abundant spores. Cultivation of the spores was carried outfollowing the procedure of Example 1.

After 15 days incubation, compounds 11, V, VI, VII and VIII wereproduced; after 30 days, compounds I, III and IV were also produced.

Three liters of lyophilized culture prepared as above were fullyextracted with ethanol in a Soxhlet extractor and concentrated tovolume. The extract was cooled slowly, when 5 grams of precipitateformed, which included the sodium salts of compounds VII and VIII. Themixture was filtered and the filtrate evaporated to dryness undervacuum. Twelve grams of product was obtained. The product was charged toa silica gel chromatographic column and eluted with a mixture of 93:7chloroformzmethanol. The following compounds were progressivelyseparated and recovered:

Compound I, 250 mg: T.L.C.***R; 0.65 (purple spot). This compound hasthe following properties: mo-

lecular formula, C d-1 0 [a]D 123 (C 0.96, methanol); M 446 (molecularions); Ultraviolet spectrum has absorption maxima at 258 mu (6 6,000ethanol). Its infrared spectra (presented as FIG. 1) shows the followingpeaks at wave lengths expressed in reciprocal centimeters when run in aKBr disc: 3,401, 3,077, 2,959, 2,924, 2,899, 2,874, 2,833, 2,817, 1,681,1,639, 1,471, 1,460, 1,439, 1,429, 1,383, 1,370, 1,344,1,325,1,292,-l,274,1,221, 1,178,l,138,1,091, 1,047, 1,015, 1,000, 961,952, 930, 909, 893, 885, 868, 856, 826, 813. This compound was solublein chloroform, methanol, ethanol and ethyl acetate. It forms anamorphous precipitate from solution in ethyl acetate.

Compound 11, mg: T.L.C.***R, 0.52 (red Spot). This compound has thefollowing propertieszmolecular formula C l-1 ,0 [0:]D -71.4 (C= 0.98,methanol), melting point ll62 C.; Ultraviolet spectrum shows noabsorption maxima between 220-400 mu; its infrared spectra (presented asFIG. 2), shows the following peaks: 3,584, 3,401, 3,086, 3,058, 2,976,2,950, 2,907, 2,865, 2,825, 2,801, 1,709, 1,681, 1,667, 1,639, 1,490,1,471, 1,460, 1,449, 1,439, 1,425, 1,412, 1,387, 1,364, 1,342, 1,330,1,311, 1,290, 1,266, 1,250, 1,230, 1,214,1,176,1,156,1,143,1,138,1,114,1,105,1,095, 1,072,1,026, 1,008, 980, 963, 951,945, 909, 886, 873, 869, 853, 839, 806, 749, 741. This compound issoluble in chloroform, methanol, ethanol and is crystallizable fromethyl acetate.

Compound III, 300 mg: T.L.C.*** R; 0.42 (purple spot). This compound hasthe following properties:- molecular formula, C,,,I-I., O [a]D 149 (C0.72, methanol); M 448; melting point -172 C.; Ultraviolet spectrumshows no absorption maxima between 220-400 mu; its infrared spectra(presented as FIG. 3), shows the following peaks: 3,534, 3,448, 3,086,3,040, 2,959, 2,950, 2,924, 2,899, 2,865, 2,817,1,689,1,68l,1,639,1,471, 1,447,1,429,1,412,1,381, 1,337, 1,206, 1,186,1,124, 1,084, 1,058, 1,047, 1,026, 1,000, 921, 81 1, 790. This compoundis soluble in chloroform, methanol and ethanol and is crystallizablefrom methanol.

Compound IV, 300 mg:T.L.C.*** R, 0.36 (brown spot). This compound hasthe following properties:- molecular formula, C d-1 0 [ctlD -113 (C0.97, methanol): M* 462; Ultraviolet spectrum has ab sorption maxima at258 mp. (e 6,000 ethanol); its infrared spectra (presented as FIG. 4),shows the following peaks: 3,401, 3,077, 2,959, 2,915, 2,874, 2,841,2,817,1,695,1,639,l,449,1,431,1,414, 1,387, 1,372, 1,342, 1,179, 1,149,1,089, 1,058, 1,000, 926, 910, 885, 862, 826. This compound is solublein chloroform, methanol, ethanol and ethyl acetate and forms anamorphous-precipitate from a solution of ethyl acetate/petroleum ether.

Compound V, 2000 mg: T.L.C.*** R,=0.34 (brown spot). This compound hasthe following properties; molecular formula, C H O [011D -32.3 (C 1.05,methanol); melting point at about 1 10 C.; Ultraviolet spectrum shows noabsorption maxima between 220-400 mu; its infrared spectra (presented asFIG. 5) shows the following peaks: 3,401, 3,086, 2,924, 2,874,2,817,1,639,1,447,1,425,1,412,1,383,1,372, 1,330,l,294,1,285,1,267,1,215,1,143,1,138,1,086,1,015, 1,000, 966, 952, 909,879, 856, 833, 830. This compound is soluble in chloroform, methanol andethanol and forms an amorphous precipitate from ethyl acetate. Y

Compound VI, 4000 mg: T.L.C.*** R,= 0.16 (green spot). This compound hasthe following properties:- molecular formula, C H O [a]D 42.7 (C 1.03,methanol); melting point at about 130 C; Ultra- EXAMPLE 5 This exampledemonstrates the preparation of compound from compound V.

violet spectrum Shows no absorption maxima between 5 one gram ofcompound V was stirred in gram of tri- 220-400 mu; its infrared spectra(presented as FIG. 6), phenylchloromethane and 4 ml of pyridine for 72hours shows the following peaks: 3,401, 3,086, 2,959, 2,907, at roomtemperature. The mixture was cooled to 0 C., 2,825, 1,637, 1,451, 1,443,1,425, 1,414, 1,383,1,372, and 6 ml of pyridine and 6 ml of aceticanhydride 1,156, 1,138, 1,070, 1,010, 966, 941, 934, 909, 883, added.After standing for 24 hours at room tempera- 832, 829, 782, 10 ture, themixture was poured into ice water. The prey cyromatografyflqlislil-GELHFluka) activated fo cipitate was collected and charged to a chromato g gf 'g ix fa m, f mmure graphic column of silica gel. The product waseluted This Compound was Soluble in methanol ethanol and withbenzenezethyl acetate (90:10). The solvent was chloroform and forms anamorphous precipitate from evaporated and grams of pr9duct 9 acetone andethyl acetate The product was then treated with acetic acid (80%) Fivegrams of the Sodium Salts reserved were at 110 C. to form thetetra-acetyl derivative. 12 grams solved in 200 ml of water and addedwith 1N hydro of product were obtained aftger chromatographicsepachloric acid up to pH 3. The resultant precipitate was h and treatdwith S reagent ($03 centrifuged and dried under vacuum. Three grams ofPyndlneDMsoftmfthylamme) to gwe P products obtained were charged to achromatographic mg trlacety] lenvatwe of! 98 Th15 compound columncontaining 100 grams of silicic acidzcelite (3:1) was treated 2 ml of 1%Sodlum methoxlde m meth' and eluted with chloroform:methano1 (90:10).Two adanol f 15 minutes at and allowed to Stand for ditional compoundswere separated and recovered. 45 mmutes at 9" temperature- 500 Mg ofCompound V11, 1400 mg: T.I ..C.** R,=0.32 (brown Pound I were Obtamedspot). This compound has the following properties:- molecular formula, Ci-1 0 [011D -85.2 (C EXAMPLE 6 1.1, methanol); melting point l92-l94 C.;Ultraviolet spectrum shows no absorption maxima between This exampledemonstrates the preparation of com- 220-400 mu; infrared spectra of itscarboxymethyl pound V from Compound Vll. ester (presented as FIG. 7),shows the following peaks: One gram of compound VII was dissolved intetrahy- 3,425, 3,086, 3,003, 2,959, 2,950, 2,933, 2,924, 2,865,drofuran and added to one gram of LiAlH, over a 1 2,849, 2,833, 1,745,1,639, 1,468, 1,449, ,437, 1,412, hour period at room temperature. Theproduct was col- 1,383, 1,350, 1,299, 1,290, 1,220, 1,142, 1,095, 1,086,lected and separated on chromatographic column. One 1,078, 1,056, 1,046,1,011, 1,010, 992, 943, 900, 881, gram of compound V was obtained. 858,847, 840, 806, 781. This compound is soluble in When the procedure ofExample 5 was repeated, 500 chloroform, methanol and ethanol and formsan amormg of compound I were obtained. phous precipitate from ethanol.

Compound VIII, 2000 mg: T.L.C.** R,=O.l8 (green EXAMPLE 7 spot). Thiscompound has the following properties:- The procedure of Example 5 wasfollowed except molecular formula, C26H4009; [0:]D -82.3 (C substituting1 gram of compound VI. 400 Mg of com- 1.13, methanol); melting pointl88l90 C.; Ultraviopound IV were obtained. let s ectrum shows noabsorption maxima between 220 100 mu; its infrared spectra (presented asFIG. 8) EXAMPLE 8 shows the following peaks: 3,546, 3,401, 3,086, 2,967,The procedure of Example 6 was repeated except 2,950, 2,899, 2,865,2,833, 2,817, 1,730, 1,667, 1,639, substituting 1 gram of compound VIII.400 Mg of com- 1,471, 1,451, 1,414, 1,389, 1,372, 1,319, 1,282, 1,227,pound IV were obtained. 1,152,1,136,1,081,1,000, 961, 951, 934, 917,893, 855, 826, 813. This compound is soluble in chloroform, EXAMPLE 9methanol and ethanol and forms an amorphous precipi- The compoundsprepared as in Example 4, were tate from ethanol. Thin layerchromatography (KIE- tested as antimycotic agents at variousconcentration, SELGEL H Fluka) activated for minutes at 1 10 and invitro on glucose-Sabourauds liquid containing eluted withchloroform-methanol-acetil acid mixture 0.5/00 chloroamphenicol. TheTable below shows the (:15:5) detection: sulphuric acid 50% at 110 for 5minimum concentration in meg/ml of test compound minutes. 55 which waseffective to inhibit the growth of test fungus.

TABLE I MINIMUM INHIBITORY CONCENTRATION IN m\c,g/ml

VI V II I IV 11 VII III Aspergillus fixmigalus 5O 50 50 12,5 25 50 50Aspergillus flavus 100 100 12,5 100 50 100 cory r n ikr 5O 50 50 1 2,5100 100 50 Penicillium mamefj"ei 12,5 25 50 12,5 25 100 50 50 jlf o lrz50 3 ,1 2 25 25 50 100 50 100 MINIMUM INHIBITORY CONCENTRATION IN mfilmlVI V II I IV II VII III Microsporum canis 12,5 3,12 25 25 50 50 50 50Trichophyton rubrum 25 I25 25 12,5 25 I 25 50 Candida albicans I00 I00I00 50 6,25 I00 50 I00 Cryptocoaus neoformans 0, I 8 0,78 0,09 6,25 0,041,56 0, I 8 50 Histoplasma capsulntum 12,5 50

EXAMPLE 10 wherein R-has the meaning given above and Y is Compounds Iand IV were tested as antibacterial agents. The minimum inhibitoryconcentration in meg/ml are given in the Table below:

TABLE II ANTIBACTERIAL SPECTRUM OF COMPOUNDS I AND IV ORGANISM MI mc mlCompound I Compound IV Staphylococcus aureus 4 5 Bacillus sublilis 3 5Escherichia coli l0 I5 MIC minimum inhibitory concentration I claim:

l. A method for producing a solution in ethanol of a mixture of thefollowing compounds:

wherein R can be hydrogen or hydroxyl and X can be a carboxyl group or aCH OH group CH() or -CI-I OH with the proviso that when Y is CH OI-l, Ris hydrogen and 'which comprises cultivating a glycoside-producingstrain of Oaspora virescens (Link) Wallr in aqueous glucose-organicdecoction media at a temperature from 10 to 25C. for a period of atleast 10 days in stationary culture, said organic decoction selectedfrom the group consisting of malt, glucose-yeast, glucose-carrot,glucose-wheat kernels, glucose-corn kernels, glucosekidney bean seeds,glucose-pea seeds and glucose-lentil seeds and having a basic pH,Iyophilizing and extracting with ethanol, cooling the ethanol solutionto form a precipitate, filtering the solution, separating the compoundsin the filtrate from each other on a chromatography column, acidifyingthe precipitate reserved from the filtering step and separating theremaining compounds on a chromatograph column.

2. A method according to claim 1 wherein the temperature is from 2325C.

3. A method according to claim 2 wherein the media is glucose-carrotdecoction.

4. A method according to claim 1 wherein the pH is from 8.6-8.8.

5. A method according to claim 3 wherein the pH is from 8.6-8.8.

6. A method according to claim 1 wherein the media is selected from thegroup consistingof malt, glucosecarrot, glucose-corn kernels andglucose-wheat kernels.

1. A METHODD FOR PRODUCING A SOLUTION IN ETHANOL OF A MIXTURE OF THEFOLLOWING COMPOUNDS:
 2. A method according to claim 1 wherein theTemperature is from 23*-25*C.
 3. A method according to claim 2 whereinthe media is glucose-carrot decoction.
 4. A method according to claim 1wherein the pH is from 8.6-8.8.
 5. A method according to claim 3 whereinthe pH is from 8.6-8.8.
 6. A method according to claim 1 wherein themedia is selected from the group consisting of malt, glucose-carrot,glucose-corn kernels and glucose-wheat kernels.